Deoxygenation enhances photosynthetic performance and increases N2 fixation in the marine cyanobacterium Trichodesmium under elevated pCO2

Effects of changed levels of dissolved O2 and CO2 on marine primary producers are of general concern with respect to ecological effects of ongoing ocean deoxygenation and acidification as well as upwelled seawaters. We investigated the response of the diazotroph Trichodesmium erythraeum IMS 101 after it had acclimated to lowered pO2 (~60 μM O2) and/or elevated pCO2 levels (HC, ~32 μM CO2) for about 20 generations. Our results showed that reduced O2 levels decreased dark respiration significantly, and increased the net photosynthetic rate by 66 and 89% under the ambient (AC, ~13 μM CO2) and the HC, respectively. The reduced pO2 enhanced the N2 fixation rate by ~139% under AC and only by 44% under HC, respectively. The N2 fixation quotient, the ratio of N2 fixed per O2 evolved, increased by 143% when pO2 decreased by 75% under the elevated pCO2. Meanwhile, particulate organic carbon and nitrogen quota increased simultaneously under reduced O2 levels, regardless of the pCO2 treatments. Nevertheless, changed levels of O2 and CO2 did not bring about significant changes in the specific growth rate of the diazotroph. Such inconsistency was attributed to the daytime positive and nighttime negative effects of both lowered pO2 and elevated pCO2 on the energy supply for growth. Our results suggest that Trichodesmium decrease its dark respiration by 5% and increase its N2-fixation by 49% and N2-fixation quotient by 30% under future ocean deoxygenation and acidification with 16% decline of pO2 and 138% rise of pCO2 by the end of this century.


Introduction
Biological N 2 fixation by marine diazotrophs is an important source of "new" usable nitrogen to phytoplankton in oligotrophic surface waters (Karl et al., 1997;Bergman et al., 2013). The N 2 -fixing cyanobacteria, Trichodesmium spp., contribute about half of the current estimate of global annual marine N 2 fixation, playing a pivotal role in global biogeochemical N/C cycles (Mahaffey et al., 2005;Sohm et al., 2011). The nitrogenase enzyme encoded by the nifHDK genes is critically sensitive to and can be inactivated by oxygen (O 2 ) (Staal et al., 2007;Hutchins and Sañudo-Wilhelmy, 2022). Most cyanobacterial N 2 -fixers can avoid O 2 damage via temporal (fixing N 2 during night) or spatial (using heterocyst) strategies. However, Trichodesmium spp. do not possess such contrivances to avoid photosynthetically evolved O 2 , since all cells of the trichomes have PSII (Lin et al., 1998;Bergman et al., 2013). It is known that Trichodesmium cells down-regulate its photosynthesis during midday to protect nitrogenase using various physiological pathways (e.g., the Mehler reaction, respiration and flavoprotein-mediated O 2 uptake) to sequestrate O 2 under light (Berman-Frank et al., 2001;Gallon, 2001;Milligan et al., 2007), which is considered as a metabolic strategy for the O 2 -sensitive diazotroph .
Dissolved O 2 (DO) levels in the oceans have been declining due to global warming, and are expected to further decline during the 21th century (Schmidtko et al., 2017;Breitburg et al., 2018). Rising global temperatures decrease the solubility of O 2 , enhancing stratification (hindering ventilation to deeper layers) of the upper mixing layers and increasing biological respiration, thus ultimately reduce DO in surface and subsurface oceans (Carstensen et al., 2014). Consequently, the oxygen-minimum zones (OMZ) have expanded horizontally and vertically along with progressive ocean deoxygenation (Stramma et al., 2008;Schmidtko et al., 2017). On the other hand, internal O 2 concentrations of Trichodesmium colonies or blooms ranged from 0 to 500 μM during dark and/or high-light periods, which is supposed to affect the nitrogenase activity (Paerl and Bebout, 1992;Eichner et al., 2019). Light stimulation of nitrogenase activity is most obvious at low O 2 concentrations, while it becomes gradually less important with increased levels of O 2 in Trichodesmium IMS 101 (Staal et al., 2007). Therefore, it is likely that the marine diazotrophs including Trichodesmium would benefit from ocean deoxygenation.
On the other hand, ocean acidification (OA) induced by continuous dissolution of anthropogenically emitted CO 2 into seawater is known to affect various phytoplankton species and ecological processes , which show positive and/or negative responses to OA under different environmental conditions (Boyd et al., 2018). Temperature, light, nutrients and UV radiation are known to modulate the influences of elevated pCO 2 , resulting in negative in oligotrophic waters but in neutral or positive effects in nutrients-replete waters . Most cyanobacteria and microalgae, including Trichodesmium, have evolved efficient CO 2 -concentrating mechanisms (CCMs) that actively uptake CO 2 and bicarbonate to increase the level of intracellular dissolved inorganic carbon for efficient photosynthesis (Giordano et al., 2005). Therefore, the energy saved by the downregulation of CCMs under elevated pCO 2 is expected to be reallocated to other metabolic processes (Kranz et al., 2010). Differential responses have also been documented in Trichodesmium. OA has been shown to promote the N 2 fixation and growth of Trichodesmium (Hutchins et al., 2007;Levitan et al., 2007;Garcia et al., 2011;Hutchins et al., 2013). However, it was also shown to result in insignificant or even negative effects on Trichodesmium, and the negative effects were more obvious under iron limitation (Shi et al., 2012;Hong et al., 2017;Zhang et al., 2019). It was suggested that the energy saved from downregulation of CCMs under elevated pCO 2 is not enough for the energy demand of increased N 2fixation rates in Trichodesmium under OA conditions (Luo et al., 2019). While these disputable findings need to be mechanistically reconciled, effects of multiple drivers on the marine diazotroph should also be examined (Wake, 2019). Recently, it was shown that reduced O 2 availability modulated the effects of elevated pCO 2 on a diatom . Combined effects of ocean deoxygenation and acidification on Trichodesmium has yet to be investigated.
Since levels of pH/pCO 2 and O 2 in the center of Trichodesmium colonies fluctuate during a diel cycle due to night respiration and daytime photosynthesis (Eichner et al., 2019), its physiological performance can be naturally influenced by the changed levels of both O 2 and CO 2 . In parallel, progressive ocean deoxygenation and acidification are covarying factors that affect marine organisms including the diazotrophs (Staal et al., 2007;Paulmier et al., 2011;Sun et al., 2022). Considering that low levels of O 2 can theoretically enhance carboxylation via decreasing oxygenation processes catalyzed by Rubisco and stimulate the activity of nitrogenase, we hypothesize that combination of ocean deoxygenation and acidification can synergistically enhance photosynthetic performance and N 2 fixation of Trichodesmium. Hence, we simulated different microenvironments by growing Trichodesmium erythraeum (T. erythraeum) strain IMS 101 under four different pO 2 /pCO 2 combinations (ambient CO 2 & ambient O 2 ; ambient CO 2 & low O 2 ; high CO 2 & ambient O 2 ; high CO 2 & low O 2 ) in order to investigate the responses of Trichodesmium. In this work, we found that reduced O 2 concentration increased both photosynthetic performance and N 2 fixation rate and thus raised the particulate organic carbon and nitrogen even under elevated pCO 2 projected for future ocean acidification by the end of this century.

Culture conditions
Trichodesmium erythraeum (strain IMS 101), originally isolated from the North Atlantic Ocean (Prufert-Bebout et al., 1993), was grown in YBCII medium without combined N prepared with autoclaved sterilized artificial seawater (Chen et al., 1996). Experiments were conducted in polycarbonate bottles under 160 μmol photons m −2 s −1 of PAR (measured by a Solar light sensor, PMA2100, United States) with a day-night cycle of 12: 12 h at 27 ± 0.5°C in an incubator (HP300G-C, Ruihua, China).
Triplicate experiments were carried out in a four treatments matrix of two levels of CO 2 (ambient and high CO 2 ) and two levels of O 2 (ambient and low O 2 ), respectively. T. erythraeum cells were maintained in the exponential growth phase by semicontinuous culture (diluted every 48 h). The chlorophyll a (Chl a) concentration was maintained within a range of 0.003-0.015 μg mL −1 (Supplementary Figure S1), so that dissolved O 2 and carbonate chemistry in the cultures were little altered. The carbonate chemistry parameters were relatively stable before and after the dilution, the pH variations were less than 0.08 units under either the HC (elevated CO 2 , 32 μM) or AC (ambient CO 2 , 12 μM) treatments, along with 12 to 19% decrease of pCO 2 before the dilutions, and dissolved O 2 concentrations varied less than 10 μM (245-255 μM) and 30 μM (60-90 μM) under AO (ambient O 2 ) and LO (low O 2 ) treatments, respectively ( buffer. Total alkalinity (TA) was measured using the Gran potentiometric titration method (Gao et al., 2018). Dissolved inorganic carbon (DIC) and other carbonate chemistry parameters (Table 1) were calculated from pH NBS and TA by using the CO2SYS software (Lewis et al., 1998).

Chl a concentration and specific growth rate
Trichodesmium erythraeum cells were gently filtered onto 25 mm glass-fiber filters (GF/F, Whatman), which was then extracted in pure methanol at 4°C for 24 h for Chl a determination. The extracts were centrifuged at 6000 g for 10 min, and the absorbances of the supernatants at 665 nm and 750 nm were measured using a UV-VIS Spectrophotometer (DU800, Beckman, United States). The Chl a concentration was calculated according to the equations Chl a (μg mL −1 ) =12.9447 × (A 665 − A 750 ) × V1/V2 (Ritchie, 2006), where V1 and V2 represent the 100% methanol volume (mL) and filtered cell volume (mL), respectively. The specific growth rates (μ, d −1 ) were determined from linear regressions of the natural log of Chl a vs. time during the exponential growth (Yi et al., 2020).

Dark respiration and net photosynthetic oxygen evolution
Dark respiration and net photosynthetic oxygen evolution were measured in the middle of the light period within 3 h using a Clarketype electrode (Hansatech, United Kingdom). Cells were harvested by filtering onto polycarbonate membrane filters (5 μm, Millipore, Germany) under gentle vacuum pressure (<0.01 MPa). These cells were then re-suspended in seawater buffered with 20 mM Tris-HCl with a final Chl a concentration of approximately 0.5 μg mL −1 . The pH levels of the Tris buffered-medium were pre-adjusted by adding hydrochloric acid or sodium hydroxide to the same levels of the cultures (pH 7.83 for HC and 8.13 for AC), and the O 2 levels were achieved by flushing the medium with pure N 2 . The resuspended cells were injected into an oxygen electrode vessel with a magnetic stirrer held in a water-jacked chamber (temperature controlled at 27°C) under the same level of light intensity. The respiration rate was estimated in darkness by covering the reaction chamber with aluminum foil. Photosynthetic O 2 evolution was determined under growth O 2 levels.

Nitrogen fixation rate
N 2 -fixation rates were determined at the midpoint of the photoperiod with the acetylene reduction assay, assuming a ratio of 4: 1 to convert ethylene production to N 2 fixation (Capone, 1993). Although one time point measurement has been able to identify changes of N 2fixation rate under different environmental conditions (Zhang et al., 2019), it may overlook other timepoint values of the N 2 -fixation during the whole light period. For determination of the N 2 -fixation rates, 5 ml of sample was added to a 13 mL vial, which was then sealed. After 1 mL of air was extracted from the headspace, 1 ml of acetylene (C 2 H 2 ) was added to each vial. All replicates were mixed well and incubated for 2 h under the same temperature and light in the incubator as the growth conditions. The ethylene production was measured using a gas chromatograph with a flame ionization detector (Clarus 580, PerkinElmer, United States).

Particulate organic carbon and nitrogen (PON)
Cell samples for particulate organic carbon (POC) and particulate organic nitrogen (PON) were collected onto pre-combusted (450°C for 6 h) GF/F filters (Whatman), and were stored at −80°C before measuring. Filters were exposed to HCl fumes to remove inorganic carbon and dried at 60°C before analysis with a CHNS elemental analyzer (vario EL cube, Elementar, Germany).

Statistical analysis
The data were presented as means of three replicates ± SD (n = 3, triplicate independent cultures). One-way ANOVA and Tukey test were performed to analyze the statistical differences among different treatments. Differences were termed significant when p < 0.05. Before performing parametric tests, data were tested for homogeneity of variance (Levene test) and normality (ShapiroWilk test).

Photosynthetic performance
Dark respiration decreased with lowered levels of O 2 , whereas net and gross photosynthetic rate increased (Figure 2). Reduced O 2 levels (from 250 to 60 μM) significantly increased net photosynthetic rate by 66% under AC (631 vs. 379 μmol O 2 mg chl a −1 h −1 , p < 0.0001) and by 89% under HC conditions (659 vs. 348 μmol O 2 mg chl a −1 h −1 , p < 0.0001), respectively ( Figure 2A). Nevertheless, no significant differences were found in the net photosynthetic rate between the AC and HC treatments (p = 0.9432) (Figure 2A), though the mean net photosynthetic rate of the cells grown under HC was higher by about 4% than that of AC under LO. Decreased O 2 levels to 60 μM (by 75%) significantly inhibited dark respiration by 40% under the AC (136 vs. 226 μmol O 2 mg chl a −1 h −1 , p = 0.0002) and 39% under the HC (172 vs. 283 μmol O 2 mg chl a −1 h −1 , p < 0.0001), respectively ( Figure 2B), reflecting that Trichodesmium would decrease its dark respiration by 9% and increase its net photosynthetic rate by 14% with 16% pO 2 decline by the end of this century (Table S1). Meanwhile, the HC-acclimated cells had higher (by 25% at AO and by 27% at LO) dark respiration rate compared to that of AC cells regardless of O 2 levels (p = 0.0031 for AO, p = 0.0278 for LO). The reduced O 2 levels significantly increased gross photosynthetic rate by 27% (767 vs. 605 μmol O 2 mg chl a −1 h −1 , p < 0.0019) under AC and by 32% (831 vs. 631 μmol O 2 mg chl a −1 h −1 , p = 0.0005) under HC, respectively ( Figure 2C). Meanwhile, HC treatments increased gross photosynthetic rate by 4% (p = 0.4844) under AO and by 8% (p = 0.1081) under LO compared to the AC-grown cells, though the difference was insignificant.

Nitrogen fixation rate
When T. erythraeum cells were grown under different O 2 levels, their N 2 fixation rates showed a negative relationship with the O 2 concentrations ( Figure 3A, r 2 = 0.88). The N 2 fixation rate increased linearly by 9% with each 10% decline in the dissolved O 2 concentration. After the Trichodesmium cells acclimated to different levels of O 2 and pCO 2 , lowering DO to about 60 μM increased the Chl a-specific N 2 fixation rate by ~139% (20.0 vs. 8.4 μmol N 2 mg chl a −1 h −1 , p < 0.0001) under AC but only by 44% (27.5 vs. 19.2 μmol N 2 mg chl a −1 h −1 , p < 0.0001) under the HC conditions ( Figure 3B), indicating obvious less enhancement by the reduced O 2 treatment under the elevated pCO 2 . The Trichodesmium cells grown in the HC treatment had higher N 2 fixation rates than those grown in the AC cultures regardless of the O 2 levels (p < 0.0001 for AO, p = 0.0001 for LO), being increased by 129% under AO and by 38% under LO, respectively. The ratio of N 2 fixation to the gross photosynthetic rate, as a proxy of the N 2 -fixation quotient (NFQ), ranged from 0.014 to 0.033 (mol N 2 : mol O 2 ) ( Figure 3C). The lowered The specific growth rates of Trichodesmium. erythraeum IMS101 grown and measured after it acclimated for 16 days (12 generations) to different combinations of pCO 2 and pO 2 (AO, ~245 μM; LO, ~60 μM; AC, ~12 μM; HC, ~32 μM. LO and HC refer to lowered pO 2 and elevated pCO 2 ). The values are indicated as the means ± SD for independent cultures (n = 6) at each treatment. The same letters above the bars indicate no significant (p < 0.05) differences among the treatments. The rates of net photosynthetic O 2 evolution (A), dark respiration (B) and gross photosynthetic O 2 evolution (C) in T. erythraeum IMS101 cells after acclimated different levels of pCO 2 and pO 2 (as shown in Figure 1) for 28-30 days (21-23 generations). Levels of pCO 2 and pO 2 are the same or within the same ranges as shown in Figure 1. The values are indicated as the means ± SD for triplicate cultures at each treatment. Different letters above the bars indicate significant (p < 0.05) differences among the treatments.
Frontiers in Microbiology 05 frontiersin.org pO 2 treatment increased the NFQ by 89% under AC (p = 0.0016) and by only 10% under HC (p = 0.5104), and the elevated pCO 2 and low pO 2 treatment increased it by 143% compared to ACAO (p < 0.0001). Based on the results in this work, we estimated that Trichodesmium would increase its N 2 -fixation by 14% and N 2 -fixation quotient by 19% under future ocean deoxygenation with 16% decline of pO 2 till 2,100 under the current pCO 2 level (Table S1).

Particulate C and N quotas
Decreased O 2 levels to about 60 μM slightly raised the cellular particulate organic carbon (POC) by 16% (p = 0.0583) and particulate organic nitrogen (PON) by 15% (p = 0.0190) in the T. erythraeum cells ISM 101 grown under the AC (Figures 4A,B). Contrastingly, under the HC conditions, the reduced O 2 concentration increased POC by 44% (p = 0.0002) and PON by 45% (p < 0.0001), respectively ( Figures 4A,B). Nevertheless, the cellular C:N ratios were about 6.8, being neither affected under the changed levels of pCO 2 (p = 0.0796) nor under the reduced levels of pO 2 (p = 0.9859) ( Figure 4C).

Discussion
In the present study, the T. erythraeum cells acclimated to lowered pO 2 and elevated pCO 2 levels showed higher N 2 -fixation quotient (ratio of N 2 fixed to gross O 2 production), being increased by 143% (with pO 2 declined bys 75%) and by 12% (with pO 2 declined by 10%) compared to ambient levels of pO 2 and pCO 2 ( Figure 5). This could be attributed to decreased dark respiration and increased photosynthetic performance as well as increased N 2 fixation under the reduced levels of pO 2 and to increased rates of dark respiration and N 2 fixation under the elevated pCO 2 (Figures 2, 3). Although both low pO 2 and high pCO 2 treatments did not significantly affect the growth rates of T. erythraeum, its cellular particulate organic carbon (POC) and nitrogen (PON) contents increased under the lowered O 2 and elevated pCO 2 levels. Our data provided the experimental evidence that reduced O 2 levels increased N 2 fixation and photosynthetic performance by stimulating the activities of nitrogenase and Rubisco ( Figure 5), enhancing POC and PON production in anoxic and/or hypoxic environments, where lowered levels of O 2 and pH (high pCO 2 ) are covarying drivers.

Responses of N 2 fixation, photosynthesis, and growth to deoxygenation
Nitrogenase, the key enzyme that catalyzes the reduction of N 2 , is instantaneously and irreversibly inactivated by O 2 via oxidative damage (Staal et al., 2007). Meanwhile, significantly decreased photosynthetic O 2 evolution coincided with increased respiratory CO 2 release during midday, that favored the N 2 fixation (Kranz et al., 2009). In the present work, reduced levels of O 2 in milieu significantly increased N 2 -fixation and photosynthesis (Figures 2, 3). Since lowered O 2 concentration increased the net photosynthetic O 2 evolution, the dissolved O 2 in the cultures increased before the continual dilutions, which, nevertheless, did not significantly alter the difference in O 2 treatments between lowered (LO) and ambient (AO) pO 2 (Table 1). Therefore, the O 2 levels (AO: 245-255 μM; LO: 60-90 μM) during the growth period could still reflect LO and AO conditions. In photosynthesis, electrons transport via the photosynthetic and respiratory transport chains must have promoted production of ATP and reductant for N 2 fixation and CO 2 assimilation in Trichodesmium (Suggett et al., 2010;Eichner et al., 2019). While no decline in net photosynthetic O 2 evolution was found with enhanced dark respiration in this study (Figure 2), higher respiration rates during the photoperiod would decrease the photosynthetic O 2 evolution in Trichodesmium (Berman-Frank et al., 2001), which must partially favored the N 2 fixation. In the present work, the rates of dark respiration accounted for 37% in AO-grown and for 18% in LO-grown cells of the gross photosynthesis ( Figures 2B,C), indicating that the role to remove O 2 by respiration was down-regulated under LO. Since the O 2 consumptions such as dark respiration were reduced under LO, and the net photosynthesis significantly increased (Figure 2), Mehler reaction must have been active in maintaining low intracellular O 2 to sustain the activity of the nitrogenase and promote energetically expensive N 2 fixation. Additionally, it is most likely that the Trichodesmium cells grown under low O 2 could have greater abundances and/or upregulated activity of the nitrogenase (Zehr et al., 1993). Such physiological responses can be responsible for increased values of the N 2 -fixation quotient under the low O 2 conditions ( Figure 3C), indicating that the N 2 -fixation efficiency increased per photosynthetically evolved O 2 or C fixed under low O 2 and high CO 2 conditions.

Combined effects of deoxygenation and acidification
Although the cells of Trichodesmium grown under HC increased their N 2 fixation under both ambient and lowered pO 2 (Figure 3), their specific growth rates were unaffected compared to the AC-grown cells (Figures 1, 2). This contradicts to some of the previously reported results (Table 2), which show either enhanced or inhibited growth rates of Trichodesmium grown under future ocean acidification conditions. The likely reason responsible for such discrepancies between these studies (Table 2) could be that different light sources (different emission spectra) regulate the diazotroph's response to elevated pCO 2 even under equal levels of PAR (Yi and Gao, 2022), though such hypothetical explanation should be based on N 2 -fixation action spectra, which has not been documented. In addition, limitation of phosphate and iron ions as well as exposure to solar UV radiation could also result in negative or insignificant effects of future ocean acidification on the N 2 -fixation of Trichodesmium (Hong et al., 2017;Yi and Gao, 2022;Zhang et al., 2022). In the present study, when the Trichodesmium cells acclimated to the acidified HC treatment with replete phosphate and iron, its photosynthesis and growth did not show significant changes, though its N 2 -fixation increased by 129% under ambient and by 38% under the reduced pO 2 , respectively ( Figure 3). In addition, the HC treatment increased its POC significantly under the LO (Figure 4), though its photosynthesis did not show significant change (Figure 2). Such inconsistency between the photosynthetic responses and the POC production could be attributed to altered photosynthetic quotients (ratios of evolved O 2 to CO 2 fixed). In the present work, gross photosynthetic rates changed less compared to the net photosynthetic rates, since dark respiration decreased when net photosynthesis increased under the different combinations of pO 2 and pCO 2 . On the other hand, the documented photosynthetic quotients of Trichodesmium ranged 1.28-2.60 (Kranz et al., 2010;Boatman et al., 2019). Therefore, photosynthetic O 2 evolution rates can hardly be taken as a proxy of C fixation, which must be responsible for the observed discrepancy between the POC production and photosynthetic O 2 evolution.
Carbon and nitrogen fixation are both energy-intensive processes that compete directly for the products of photosynthesis (Berman-Frank et al., 2001;Hutchins et al., 2007). Previously, the enhancement of N 2 fixation as well as growth rate of Trichodesmium at elevated pCO 2 levels has been considered as an indirect effect resulting from alleviation of C-limitation of CO 2 fixation (Hutchins et al., 2007) or attributed to saved energy from down-regulation of CCMs (Table 2). However, Shi et al. (2012) and Hong et al. (2017) showed that the N 2 fixation rate of T. erythraeum significantly decreased under the elevated pCO 2 projected for future acidification, which was attributed to reduced efficiency of nitrogenase, although the expression of the nitrogenase was enhanced in terms of proteomic responses (Zhang et al., 2019;Wen et al., 2022). Our data did not show significant effects of the elevated pCO 2 on the growth rate and photosynthesis in T. erythraeum, but its N 2 fixation rate and POC/PON were enhanced (Figures 2-4). It is likely that the energy saved from downregulation of CMMs under HC was not enough to bring significant increase of net photosynthesis and growth rate (Luo et al., 2019). Considering that phytoplankton can benefit from the elevated pCO 2 under light but suffer from the acidic stress in darkness, resulting in daytime enhanced and nighttime suppressed growth rates (Qu et al., 2021), the net effects of the HC treatment on growth should be holistically considered for the diel cycle and availability of nutrients (Table 2). We suggest that extra energy required to cope with the acidic stress under HC could be provided via increased dark respiration (Figure 2), compensating for the insufficient energy supply from the downregulation of CCMs in Trichodesmium cells grown under HC. In the present work, lowered levels of O 2 enhanced photosynthesis and POC production of Trichodesmium (Figures 2, 4). This could be attributed to suppressed respiration (Figure 2), which enables the cells to save more POC ( Figure 4A).

Discrepancy between the N 2 fixation and PON production
Since one time point measurement might have overlooked daily N 2 fixation, the rates of N 2 fixation in this work could hardly be approximated to that of PON production (Figures 3, 4). When the N 2 fixation rate was integrated for the daytime, it turned out that PON production rate (Supplementary Figure S3) was higher than the integrated N 2 fixation per day, which was about one third of the PON production rates. Similar discrepancy was also reported by Zhang et al. (2019), whose daily PON production rate was about 3 times the daily integrated N 2 fixation. In the present work, increased proportion (15%) of PON is much less than the The particulate organic carbon (POC) (A), particulate organic nitrogen (PON) per chl a (B), and C:N ratio (mol: mol) (C) in T. erythraeum IMS101 grown and acclimated under the different levels of O 2 and CO 2 (see Figure 1) for 36 days (27 generations (Figures 3, 4), such inconsistency could be contributed to the fact that PON production reflects how much new fixed N is ultimately incorporated into the cells which covers several generations, while N 2 fixation rate measured within 2 h during the midday could only reflect the periodical performance during light period. Future studies should examine the diurnal variations of the N 2 fixation to look into the diel or diurnal responses. Meanwhile, increased availability of CO 2 (Kranz et al., 2010;Hutchins et al., 2015) and elongated light period (Cai and Gao, 2015) could alter the maximal rate of N 2 fixation from the mid-light period, the low O 2 treatment might have led to different levels of N 2 fixation rates at different time points during the Illustration of the effects of ocean deoxygenation and acidification on Trichodesmium erythraeum, which is suggested to decrease its dark respiration by 5% and to increase its N 2 -fixation by 49% and N 2 -fixation quotient (NFQ, ratios of N 2 fixed per C fixed) by 30% with 16% decline of pO 2 and 138% rise of pCO 2 by the end of this century. Frontiers in Microbiology 08 frontiersin.org light period, which could also be responsible for the inconsistency. On the other hand, since the cells can release 10 to 50% of the fixed N as dissolved organic N (DON) and / or NH 4 + into seawater (Mulholland et al., 2004;Konno et al., 2010;Lu et al., 2018), the proportional increase of PON could be less. The exudation of "new-fixed" N may be further increased at elevated pCO 2 (Hutchins et al., 2007), which is mirrored in our study in that the elevated pCO 2 enhanced the N 2 fixation but not changed the cellular PON under the ambient O 2 level (Figures 3, 4).

Implications for future ocean deoxygenation and acidification
The global oxygen loss has been suggested to be about 2% of the total ocean inventory per decade since 1960 (Schmidtko et al., 2017) and the O 2 concentration of ocean has been predicted to decline to about 200 μmol L −1 by the end of the century (−5 μmol kg −1 per decade) (Breitburg et al., 2018). In addition, upwelling-induced hypoxia events have been shown in sunlit layers in time-series observations (Gireeshkumar et al., 2017), and typhondriven mixing would churn deep seawater of low O 2 and high CO 2 to surface layer. Therefore, diazotrophs and other phytoplankton are inevitably exposed to extreme low O 2 and high CO 2 conditions. This study provided the experimental evidence that combination of lowered pO 2 and pH along with elevated pCO 2 enhanced the photosynthesis and N 2 -fixation of Trichodesmium. Under the future scenarios of ocean deoxygenation and acidification, as estimated in the present work, Trichodesmium would decrease its dark respiration by 5% and increase its N 2 -fixation by 49% and N 2 -fixation quotient by 30% with 16% decline of pO 2 and 138% rise of pCO 2 by the end of this century (Supplementary Table S1; Figure 5). Such estimation might overestimate the changed percentage, since increased POC and PON only accounted for 10% and by 8%, respectively ( Figure 4). As increased temperature would also enhance iron use efficiency (Jiang et al., 2018) and stimulate dark respiration and then favors N 2 -fixation, decreased O 2 availability along with ocean warming can further enhance the activity of the nitrogenase and increased N 2 -fixation of Trichodesmium, as long as ocean warming does not surpass the thermal tipping point for its growth.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions
KG and HL designed the experiment, and analyzed the data and wrote and improved the manuscript. HL carried out the experiment. All authors contributed to the article and approved the submitted version.

Funding
This study was supported by National Natural Science Foundation of China (nos. 41721005, 41720104005, and 41890803).